Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citations for these references may be found at the end of this application, preceding the sequence listing and the claims.
The ob gene has recently been cloned and shown to encode a 146 amino acid protein called leptin which is secreted into the blood exclusively by white fat adipocytes. The leptin receptor has also recently been cloned and shown to exist as several splice variants. The functional splice variant (Ob-Rb) is present in several tissues including hypothalamus, adipocytes and kidney. The short form splice variant (Ob-Ra) has a more ubiquitous tissue distribution and is more abundant than the functional splice variant. The role of Ob-Ra is unknown but this splice variant may serve as a leptin transport system in choroid plexus, kidney and perhaps also the lung. A third major splice variant (Ob-Re) has also been described in mice which encodes only the extracellular domain of the receptor, giving rise to a soluble protein in the circulation which may function there as a leptin binding/buffering system.
Circulating leptin acts as an antiobesity agent by restraining appetite and altering metabolic processes to burn fat. The hypothalamus appears to be the major target tissue for the hormone since leptin receptors are present there and intracerebroventricular injection of leptin leads to a reduction of food intake. Recent evidence suggests that at least part of the reduction in food intake produced by leptin may be due to a decrease in central neuropeptide Y. It is unclear whether the changes in metabolism produced by leptin are mediated by actions only on the brain or also involve direct effects upon peripheral tissues. Although the mechanism of action has not been fully elucidated, leptin may be the long sought after satiety factor released from the periphery (i.e. the adipocytes) to regulate long-term body weight. As body weight and fat mass increase, more leptin is secreted which may inhibit appetite and increase metabolism to bring the fat mass back to a certain set point. The incapacity to express a functional leptin is the cause of obesity in the ob/ob mouse. Defects in the functional leptin receptor such as those found in the db/db mouse and the fa/fa rat are responsible for the obesity observed in these animal models.
The observation that obese animals and man, although having high plasma leptin levels remain overweight, may suggest the development of xe2x80x98resistancexe2x80x99 to the actions of leptin. This may occur at the level of the brain and be due to saturation of the leptin uptake system. Alternatively, xe2x80x98resistancexe2x80x99 may be due to the presence of a circulating binding protein which by buffering leptin might reduce its actions.
The single gene defects of rodents described above may play only a minor role in human obesity. However, leptin is present in man, thus, pharmacological stimulation of the leptin pathway has the potential to reduce body weight in man by inhibiting food intake and diminishing the size of the body fat stores. Such therapeutic interventions could be achieved either by enhancing leptin release from the adipocytes, preventing the breakdown or clearance of leptin, preventing interaction of leptin binding to the soluble binding protein, by administration of leptin mimics or by stimulating events downstream of the leptin receptor. On the other hand, pharmacological inhibition of leptin action or production may have the potential to increase food intake and body weight in man. Evidence to support this comes from the ob/ob and db/db mouse and the fa/fa rat in which the actions of leptin are not apparent. These animals are obese and hyperphagic. Applicants now report the isolation of a novel human Ob-Re receptor, referred to herein as xe2x80x9chOb-Rexe2x80x9d or the xe2x80x9cpolypeptide.xe2x80x9d This discovery provides a novel approach to the treatment of eating disorders, both by therapeutic administration of the soluble human Ob-Re receptor to subjects suffering from such disorders, and through the use of heterologous expression systems to develop high-affinity compounds that could serve as therapeutic agents for such disorders.
This invention is directed to an isolated nucleic acid which encodes a polypeptide comprising the amino acid sequence shown in FIG. 5 (Seq. I.D. No. 10) or a polypeptide having a sequence which varies therefrom by no more than 15 amino acids, such amino acid variations not involving amino acid positions 799-804 and not changing the functional properties of the polypeptide.
This invention is additionally directed to a nucleic acid which comprises the nucleic acid of above linked to a nucleic acid encoding a polypeptide corresponding to an artificial transmembrane region of a receptor which is not an Ob receptor.
This invention is additionally directed to a nucleic acid which comprises the nucleic acid of above linked to nucleic acid encoding a polypeptide corresponding to an artificial intracellular domain of a receptor which is not an Ob receptor.
This invention is additionally directed to purified polypeptides encoded by the nucleic acid of this invention.
This invention is additionally directed to vectors comprising the nucleic acid of this invention.
This invention is additionally directed to cells comprising the vector of this invention.
This invention is additionally directed to a membrane preparation isolated from the cell of this invention.
This invention is additionally directed to a nucleic acid probe comprising at least 15 nucleotides, which probe has a unique sequence corresponding to a sequence present within (a) the nucleic acid sequence from nucleotide number 2395 through nucleotide number 2412 of FIG. 4 (Seq. I.D. No. 9) or (b) a reverse complement thereof.
This invention is additionally directed to an antisense oligonucleotide having a unique sequence corresponding to a sequence present within (a) the nucleic acid sequence from nucleotide number 2395 through nucleotide number 2412 of FIG. 4 (Seq. I.D. No. 9) or (b) a reverse complement thereof.
This invention is additionally directed to an antibody capable of specifically binding to the polypeptide containing at least a unique sequence corresponding to a sequence present within the amino acid sequence from amino acid number 799 through amino acid number 804 of FIG. 5 (Seq. I.D. No. 10).
This invention is additionally directed to a pharmaceutical composition comprising an amount of the oligonucleotide effective to reduce expression of a polypeptide and a pharmaceutically acceptable carrier.
This invention is additionally directed to a pharmaceutical composition which comprises an amount of the antibody effective to block binding of a ligand to the polypeptide and a pharmaceutically acceptable carrier.
This invention is additionally directed to a transgenic nonhuman mammal expressing a nucleic acid of this invention.
This invention is directed to a process for identifying a chemical compound which specifically binds to a polypeptide of this invention, which comprises contacting the polypeptide with the compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the polypeptide.
This invention is additionally directed to a process involving competitive binding for identifying a chemical compound which specifically binds to a polypeptide of this invention which comprises separately contacting the polypeptide, with both the chemical compound and a second chemical compound known to bind to the polypeptide, and with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting specific binding of the chemical compound to the polypeptide, a decrease in the binding of the second chemical compound to the polypeptide in the presence of the chemical compound indicating that the chemical compound binds to the polypeptide.
This invention is additionally directed to a process for identifying a chemical compound which specifically binds to a polypeptide encoded by a nucleic acid of this invention, which comprises contacting cells containing DNA encoding and expressing on the cell surface the polypeptide, with the compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the polypeptide.
This invention is directed to a process for identifying a chemical compound which specifically binds to a polypeptide encoded by a nucleic acid of this invention, which comprises contacting a membrane fraction from a cell extract of cells containing DNA encoding and expressing on their cell surface the polypeptide, with the compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the polypeptide.
This invention is directed to a process involving competitive binding for identifying a chemical compound which specifically binds to a polypeptide encoded by a nucleic acid of this invention, which comprises separately contacting cells expressing on their cell surface the polypeptide, with both the chemical compound and a second chemical compound known to bind to the polypeptide, and with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting specific binding of the chemical compound to the polypeptide, a decrease in the binding of the second chemical compound to the polypeptide in the presence of the chemical compound indicating that the chemical compound binds to the polypeptide.
This invention is directed to a process involving competitive binding for identifying a chemical compound which specifically binds to a polypeptide encoded by a nucleic acid of this invention, which comprises separately contacting a membrane fraction from a cell extract of cells expressing on their cell surface the polypeptide, with both the chemical compound and a second chemical compound known to bind to the polypeptide, and with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting specific binding of the chemical compound to the polypeptide, a decrease in the binding of the second chemical compound to the polypeptide in the presence of the chemical compound indicating that the chemical compound binds to the polypeptide.
This invention is directed to a method of screening a plurality of chemical compounds not known to bind to a polypeptide encoded by a nucleic acid of this invention to identify a compound which specifically binds to the polypeptide, which comprises:
(a) contacting cells transfected with and expressing DNA encoding the polypeptide with a compound known to bind specifically to the polypeptide;
(b) contacting the preparation of step (a) with the plurality of compounds not known to bind specifically to the polypeptide, under conditions permitting binding of compounds known to bind the polypeptide;
(c) determining whether the binding of the compound known to bind to the polypeptide is reduced in the presence of the plurality of compounds, relative to the binding of the compound in the absence of the plurality of compounds; and if so
(d) separately determining the binding to the polypeptide of each compound included in the plurality of compounds, so as to thereby identify the compound which specifically binds to the polypeptide.
This invention is directed to a method of screening a plurality of chemical compounds not known to bind to a polypeptide of this invention to identify a compound which specifically binds to the polypeptide, which comprises:
(a) preparing a cell extract or cell supernatant from cells transfected with and expressing DNA encoding the polypeptide and contacting the cell extract or cell supernatant with a compound known to bind specifically to the polypeptide;
(b) contacting the preparation of step (a) with the plurality of compounds not known to bind specifically to the polypeptide, under conditions permitting binding of compounds known to bind the polypeptide;
(c) determining whether the binding of the compound known to bind to the polypeptide is reduced in the presence of the compounds, relative to the binding of the compound in the absence of the plurality of compounds; and if so
(d) separately determining the binding to the polypeptide of each compound included in the plurality of compounds, so as to thereby identify the compound which specifically binds to the polypeptide.
This invention is directed to a process for determining whether a chemical compound is an Ob receptor agonist which comprises contacting cells transfected with and expressing DNA of this invention with the compound under conditions permitting the activation of the Ob receptor, and detecting an increase in Ob receptor activity, so as to thereby determine whether the compound is an Ob receptor agonist.
This invention is directed to a process for determining whether a chemical compound is an Ob receptor agonist which comprises preparing a cell extract from cells transfected with and expressing DNA of this invention, isolating a membrane fraction from the cell extract, contacting the membrane fraction with the compound under conditions permitting the activation of the Ob receptor, and detecting an increase in Ob receptor activity, so as to thereby determine whether the compound is an Ob receptor agonist.
This invention is directed to a process for determining whether a chemical compound is an Ob receptor antagonist which comprises contacting cells transfected with and expressing DNA of this invention with the compound in the presence of a known Ob receptor agonist, under conditions permitting the activation of an Ob receptor, and detecting a decrease in Ob receptor activity, so as to thereby determine whether the compound is an Ob receptor antagonist.
This invention is directed to a process for determining whether a chemical compound is an Ob receptor antagonist which comprises preparing a cell extract from cells transfected with and expressing DNA of this invention, isolating a membrane fraction from the cell extract, contacting the membrane fraction with the ligand in the presence of a known Ob receptor agonist, under conditions permitting the activation of the Ob receptor, and detecting a decrease in Ob receptor activity, so as to thereby determine whether the compound is an Ob receptor antagonist.
This invention is directed to a pharmaceutical composition comprising an effective amount of a polypeptide of this invention and a pharmaceutically acceptable carrier.
This invention is directed to a method for determining whether a compound modulates leptin activity which comprises:
(a) administering to an animal a polypeptide of this invention and measuring the amount of food intake, metabolic, or body weight changes in the animal;
(b) administering to a second animal both the polypeptide and the compound, and measuring the amount of food intake, metabolic, or body weight changes in the second animal; and
(c) determining whether the amount of food intake, metabolic, or body weight change is altered in the presence of the compound relative to the amount of food intake, metabolic, or body weight change in the absence of the compound, so as to thereby determine whether the compound modulates leptin activity.
This invention is directed to a method of screening a plurality of compounds to identify a compound which modulates leptin activity which comprises:
(a) administering to an animal a polypeptide of this invention and measuring the amount of food intake, metabolic, or body weight changes in the animal;
(b) administering to a second animal the polypeptide and at least one compound of the plurality of compounds and measuring the amount of food intake, metabolic, or body weight changes in the animal;
(c) determining whether the amount of food intake, metabolic, or body weight change is altered in the presence of at least one compound of the plurality relative to the amount of food intake, metabolic, or body weight change in the absence of at least one compound of the plurality, and if so;
(d) separately determining whether each compound modulates leptin activity according to the method of this invention, so as to thereby identify a compound which modulates leptin activity.
This invention is directed to a method of treating an abnormality in a subject, wherein the abnormality is alleviated by modulating the activity of leptin in the subject, which comprises administering to a subject an amount of the pharmaceutical composition of this invention effective to modulate the activity of leptin in the subject, thereby treating the abnormality in the subject.
This invention is directed to a method of modulating feeding behavior or metabolism of a subject which comprises administering to the subject an amount of a polypeptide of this invention effective to modulate the feeding behavior or metabolism of the subject so as to thereby modulate feeding behavior or metabolism of the subject.
This invention is directed to a method of modulating feeding behavior or metabolism of a subject which comprises administering a polypeptide of this invention and a compound which binds to the Y5 receptor, the amount of such polypeptide and compound being effective to modulate the feeding behavior or metabolism of the subject.
This invention is directed to a method of modulating feeding behavior or metabolism in a subject which comprises administering to the subject an amount of a compound which binds to a polypeptide of this invention effective to alter the activity of leptin in the subject, so as to thereby modulate feeding behavior or metabolism of the subject.
This invention is directed to a method of modulating feeding behavior or metabolism of a subject which comprises administering a compound which binds to a polypeptide of this invention and a second compound which binds to the Y5 receptor, the amount of the first compound and the second compound being effective to modulate the feeding behavior or metabolism of the subject.
This invention is directed to a method of detecting expression of a polypeptide of this invention by detecting the presence of mRNA coding for the polypeptide which comprises obtaining total mRNA from the cell and contacting the mRNA so obtained with the nucleic acid probe of this invention under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of the polypeptide by the cell.
This invention is directed to a method of detecting the presence of a polypeptide which comprises contacting the cell or cell supernatant with the antibody of this invention under conditions permitting binding of the antibody to the polypeptide, detecting the presence of the antibody bound to the cell or cell supernatant, and thereby detecting the presence of a polypeptide.
This invention is directed to a method of determining the physiological effects of varying levels of activity of polypeptides which comprises producing a transgenic nonhuman mammal of this invention whose levels of polypeptide activity are varied by use of an inducible promoter which regulates polypeptide expression.
This invention is directed to a method of determining the physiological effects of varying levels of activity of polypeptides which comprises producing a panel of transgenic nonhuman mammals of this invention each expressing a different amount of polypeptide.
This invention is directed to a method for diagnosing a predisposition to a disorder associated with the activity of a specific polypeptide allele which comprises:
(a) obtaining DNA of subjects suffering from the disorder;
(b) performing a restriction digest of the DNA with a panel of restriction enzymes;
(c) electrophoretically separating the resulting DNA fragments on a sizing gel;
(d) contacting the resulting gel with a nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a polypeptide and labeled with a detectable marker;
(e) detecting labeled bands which have hybridized to the nucleic acid of this invention labeled with a detectable marker to create a unique band pattern specific to the DNA of subjects suffering from the disorder;
(f) preparing DNA obtained for diagnosis by steps a-e; and
(g) comparing the unique band pattern specific to the DNA of subjects suffering from the disorder from step e and the DNA obtained for diagnosis from step f to determine whether the patterns are the same or different and to diagnose thereby predisposition to the disorder if the patterns are the same.
This invention is directed to a method of preparing the purified polypeptide of this invention which comprises:
(a) inducing cells to express the polypeptide;
(b) recovering the polypeptide from the induced cells; and
(c) purifying the polypeptide so recovered.
This invention is directed to a method of preparing the purified polypeptide of this invention which comprises:
(a) inserting nucleic acid encoding the polypeptide in a suitable vector;
(b) introducing the resulting vector in a suitable host cell;
(c) placing the resulting cell in suitable condition permitting the production of the isolated polypeptide;
(d) recovering the polypeptide produced by the resulting cell; and
(e) purifying the polypeptide so recovered.